In recent years, various important products have been produced on a commercial scale using microorganisms or recombinant microorganisms. In many cases, those products are produced and accumulated within microbial cells and, for recovering them, a step of decomposing or lysing the microbial cells is generally required. One of the problems encountered in this step is that when cells are decomposed or lysed, the viscosity of the solution increases as a result of extracellular liberation of nucleic acids. When released into a solution, nucleic acids get unfolded and form a network structure. This causes a marked increase in the viscosity of the cell lysation product and, for example, the whole solution becomes a gel and, accordingly, the solid/liquid separation becomes very difficult to make. Thus, the nucleic acid-induced viscosity increase can become a serious problem in the step of product recovery.
In the art, attempts have so far been made to chemically or physically degrade or degrade nucleic acids.
As for the method of chemically treating microbial cells (hereinafter sometimes referred to also as “microbes” or “cells”), U.S. Pat. No. 5,110,980 discloses a method of product recovery which comprises treating a suspension of cells with a large amount of hypochlorous acid to thereby chemically decompose and solubilize cell-constituting components other than the desired product. Although this method enables efficient degradation of nucleic acids, it is necessary, for obtaining a highly pure product, to carry out the hypochlorous acid treatment at a microorganism concentration of only 1%. Therefore, a large amount of hypochlorous acid is required and, further, the odor of chlorine remains in the product and a reduction is caused in molecular weight of the product; hence, this method is not suited for practical use.
In Japanese Kokai Publication Sho-58-212792, sodium hypochlorite is presented as a substance inducing autodigestion of cell walls, not nucleic acids. However, there is no detailed description or example of the relevant invention, hence the invention can hardly be said to have been completed.
Japanese Kohyo Publication Hei-08-502415 discloses a method comprising degradation of nucleic acids liberated upon surfactant treatment of cells with a peroxide. At room temperature, it takes 16 hours to chemically degrade those nucleic acids using hydrogen peroxide as the peroxide. The amounts of the surfactant and hydrogen peroxide are relatively large. Accordingly, this method is disadvantageous from the industrial production viewpoint.
Japanese Kokoku Publication Hei-04-61638 shows a recovery method according to which heat treatment is used in combination with an enzyme and a surfactant. This method is a method of recovering a polyhydroxyalkanoate (hereinafter referred to as “PHA” for short) which comprises preliminarily heating a microbial cell suspension at 100° C. or above to degrade nucleic acids and then decomposing cell constituents other than the PHA using any of various enzymes, followed by PHA recovery. However, the molecular weight of the PHA is markedly reduced upon heating at 100° C. or above, so that the product obtained is no longer suited for practical use.
In addition, there are the following methods of disrupting cells disclosed: the method comprising heating a PHA-containing microbial cell suspension at 50° C. or above under strong acidity conditions, namely at a pH below 2, followed by PHA separation (Japanese Kokai Publication Hei-11-266891), the method comprising adding an alkali to cells, then heating the resulting mixture at 80° C. and, after 1 hour of stirring, recovering the polymer by centrifugation (Japanese Kokai Publication Hei-07-31487), the method comprising carrying out high pressure disruption at 70° C. (Japanese Kokai Publication Hei-07-31488), and the method comprising adding an alkali and then carrying out high pressure disruption at 70° C. or above (Japanese Kokai Publication Hei-07-31489). While these methods cause disruption of nucleic acids as well, the acid, alkali, temperature and other conditions are severe, so that there arises the possibility of the decomposition of the product proceeding.
Thus, it is to be understood that, in product recovery from cells after cultivation, it is very difficult to efficiently disrupt nucleic acids in microbial cells without causing the product to be decomposed. Therefore, any process involving cell decomposition or lysis requires an effective method for degradation of nucleic acids so that the subsequent product recovery step may be carried out effectively or actually perfectly.